In various noxious compounds such as pesticides or toxins, and biologically active important substances such as hormones, there are many compounds which are sparingly soluble in water, but soluble in oil. Hitherto, when the qualitative and quantitative determination of a hydrophobic (water-sparingly-soluble) but lipophilic compound is carried out, the hydrophobic but lipophilic compound to be examined was extracted from a sample by an appropriate organic solvent and purified, and then various instrumental analyses or the like were performed. These procedures were cumbersome and time-consuming in comparison with the case of hydrophilic (water-soluble) compounds which can be directly qualitatively and quantitatively assayed. For example, when assaying okadaic acid which is a toxin contained in marine products, in particular, a diarrheal shellfish poison contained in bivalves such as scallops, the extraction was performed by acetone, ether, ethyl alcohol or methyl alcohol, and the extract was concentrated if necessary, and the assay was carried out by high performance liquid chromatography.
Further, when assaying the above hydrophobic but lipophilic compounds by the immunological method, it was necessary, prior to the determination, to extract the sample possibly containing the substance to be examined by an organic solvent selected in accordance with the extent of the oil solubility of the substance in question. These procedures were cumbersome and time-consuming in comparison with the case of hydrophilic compounds. Further, if there remained the organic solvent used for the extraction, the immunoreaction would be inhibited and thus the reaction would not proceed, or extremely inaccurate results would be obtained in the assay, depending on the concentration of the remaining solvent. To remedy such an inaccurate immunoassay, the organic solvent extract of the lipophilic compound might be diluted with water to lower the concentration of the organic solvent to a concentration where the immunoreaction proceeds accurately (for example, 40% or less). However, even if the immunoreaction could proceed accurately, the solubility of the lipophilic compound per se would be reduced and thus an accurate assay still could not be performed.
Therefore, one of the objects of the present invention is to provide a means enabling swift and specific assay of a hydrophobic but lipophilic compound by immunological means using an antibody.
In the meanwhile, there exist three kinds of diarrheal shellfish poisons; okadaic acid, dinophysistoxin-1, and dinophysistoxin-3 (that is, 7-O-acyl-dinophysistoxin-1). Okadaic acid is a lipophilic compound produced by sponges belonging to Halichondria (Halichondria-okadai and Halichondria-meranodocia), and dinophysistoxin-1 is a lipophilic compound produced by Dinophysis fortii. Further, dinophysistoxin-3 is a lipophilic compound produced by converting dinophysistoxin-1 inside the shellfish. These compounds accumulate in the mesenteron glands of edible bivalves in certain seasons and regions to make the shellfish toxic. The diarrheal shellfish poison is the second most frequent type of food poisoning after blowfish in terms of number of outbreaks, but is the number one in terms of the number of victims, and therefore is a major problem in food sanitation.
Hitherto, the measurement of lethal activity using mice is adopted as the official method of examination of diarrheal shellfish poisons, but there were problems in terms of the management of the animals, the sensitivity of detection, the precision, and the specificity. On the other hand, attempts have been made to develop techniques aimed at performing the above examination with a high sensitivity, in a simple manner, and in a short time.
For example, Japanese Unexamined Patent Publication (Kokai) No. 1-96199 discloses a monoclonal antibody specific to a group of the okadaic acids and a process for production of the monoclonal antibody. However, this antibody reacts in a specific manner with okadaic acid and dinophysistoxin-1 among the diarrheal shellfish poisons, but does not react with dinophysistoxin-3. Therefore, it is unable to detect or measure the latter. Further, the above-mentioned Japanese Unexamined Patent Publication (Kokai) No. 1-96199 includes no description relating to obtaining a monoclonal antibody which can maintain its activity in the presence of an organic solvent and no suggestion thereabout.
The present inventors paid attention to the facts that the food poisoning by shellfish poisons is caused mainly by dinophysistoxin-3 in Japan and that all of the above shellfish poisons are lipophilic compounds, and thus considered that it is inevitable to use organic solvents for extracting the shellfish poison components from a sample and it is desirable to carry out an immunoreaction in the presence of an organic solvent for simplification of the procedures. Therefore, the present inventors engaged in the study to solve these problems, and successfully discovered a mouse monoclonal antibody which is specific to the main components of diarrheal shellfish poisons, that is, okadaic acid, dinophysistoxin-1 and dinophysistoxin-3 and which is resistant to organic solvents, and, in addition, discovered that when this monoclonal antibody is used, it is possible to immunologically determine diarrheal shellfish poisons quickly in a specific manner, even in the presence of an organic solvent. Therefore, the present invention relates also to a monoclonal antibody, a hybridoma secreting the monoclonal antibody, and a method of determination using the monoclonal antibody.